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Divisions of Pulmonary and Critical Care Medicine and Allergy and Clinical Immunology, Johns Hopkins School of Medicine, Baltimore, Maryland 21224
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Recent studies demonstrate
that endothelin-1 (ET-1) constricts human pulmonary arteries (PA). In
this study, we examined possible mechanisms by which ET-1 might
constrict human PA. In smooth muscle cells freshly isolated from these
arteries, whole cell patch-clamp techniques were used to examine
voltage-gated K+ (KV) currents. KV
currents were isolated by addition of 100 nM charybdotoxin and were
identified by current characteristics and inhibition by 4-aminopyridine
(10 mM). ET-1 (10
8 M) caused significant inhibition of
KV current. Staurosporine (1 nM), a protein kinase C (PKC)
inhibitor, abolished the effect of ET-1. Rings of human intrapulmonary
arteries (0.8-2 mm OD) were suspended in tissue baths for
isometric tension recording. ET-1-induced contraction was maximal at
108 M, equal to that induced by KV channel
inhibition with 4-aminopyridine, and attenuated by PKC inhibitors.
These data suggest that ET-1 constricts human PA, possibly because of
myocyte depolarization via PKC-dependent inhibition of KV.
Our results are consistent with data we reported previously in the rat,
suggesting similar mechanisms may be operative in both species.
lung; protein kinase C; vascular smooth muscle; pulmonary arterial smooth muscle cells
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOTHELIN (ET)-1 is a 21-amino acid peptide secreted by the vascular endothelium. After its release from the endothelium, ET-1 contracts smooth muscle by binding to either type A or type B endothelin receptors, both of which are abundantly present in the pulmonary vasculature (21). ET-1-induced constriction is well characterized in the pulmonary vasculature of the cat, rat, guinea pig, dog, and rabbit (5, 7, 17, 19, 22, 23, 25, 34). Recently, ET-1 was found to cause constriction in human pulmonary arteries (6, 15, 27, 31). Understanding the mechanisms by which ET-1 causes constriction in the human pulmonary vasculature is important, since a growing body of evidence implicates ET-1 as an important modulator of pulmonary vascular tone and suggests that ET-1 may be involved in the pathogenesis of hypoxic pulmonary hypertension. For example, ET-1 levels are markedly increased during chronic hypoxia (10, 12, 14), and ET receptor antagonists prevent and partially reverse the development of pulmonary hypertension secondary to chronic hypoxia (8, 10, 12). Furthermore, pulmonary arteries appear to be hyperresponsive to exogenous ET-1 after chronic exposure to hypoxia (13, 24, 27, 31).
In the lung, the mechanism by which ET-1 induces arterial smooth muscle cell contraction appears to be heavily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i) resulting from Ca2+ influx through voltage-gated Ca2+ channels (17, 25, 36) and Ca2+ release from intracellular stores (5, 19, 36). In rats, we have previously demonstrated that ET-1-induced Ca2+ influx occurs through voltage-gated Ca2+ channels secondary to membrane depolarization, which is due, in part, to protein kinase C (PKC)- and/or phospholipase C (PLC)-dependent inhibition of voltage-gated K+ (KV) channels (35, 36).
Several subtypes of K+ channels have been pharmacologically and molecularly identified in pulmonary arterial smooth muscle cells (PASMCs), including ATP-sensitive K+ (KATP), Ca2+-activated K+ (KCa), and KV channels (2, 3, 30, 35, 37-39). In this cell type, KV channels are the main regulators of membrane potential, since inhibitors of these channels, but not KCa or KATP channels, cause depolarization and increased [Ca2+]i (2, 35, 38). In cultured human PASMCs, ET-1 can activate and inhibit KCa channels, depending on the concentration (31). Interestingly, ET-1 was found to have no effect on KV channels in these cells (31). In culture, however, K+ channel and ET receptor distribution might be altered; thus, the effect of ET-1 on K+ channels in cultured PASMCs may not accurately reflect the effects of ET-1 on K+ channels in freshly isolated cells. Therefore, in this study, we used human pulmonary arterial segments and freshly isolated human PASMCs to 1) verify that ET-1 constricts human pulmonary arteries, 2) characterize KV currents, 3) determine whether ET-1 inhibits KV channels, and 4) determine whether the effect of ET-1 on KV channels is PKC dependent.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tissue Preparation
Human lung tissue was obtained from 18 anonymous organ donors (supplied by the International Institute for the Advancement of Medicine, Exton, PA, or the Anatomical Gift Foundation, Woodbine, GA). Organ donor specimens were mainly obtained from victims of head trauma or cerebral vascular accidents. The donors were men (10) and women (8) with an average age of 28 ± 3 yr (range 10-57 yr). The lungs were macroscopically normal; lungs from donors with documented pulmonary pathology were not used. Donor organs were placed in cooled (4°C) MEM and transferred to the laboratory within 24 h. Upon arrival, tissues were immediately placed in cold physiological saline solution (PSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH.Isolation of PASMCs
Single smooth muscle cells were obtained using methods previously described (35). Briefly, intrapulmonary arteries (800-2,000 µM OD) from the lower right lobe were dissected free of connective tissue and opened, and the lumen was gently scraped with a cotton swab to remove the endothelial cells. The arteries were placed in cold PSS for 30 min and then transferred to reduced Ca2+ PSS (20 µM CaCl2) at room temperature for 20 min. The tissue was enzymatically digested for 20 min at 37°C in reduced Ca2+ PSS containing 5.5 mg/ml collagenase, 0.6 mg/ml papain, 2 mg/ml BSA, and 1 mM dithiothreitol. After digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free PSS. The cell suspension was transferred to glass coverslips for study.Membrane Current Measurements
Myocytes were continuously superfused with PSS. Membrane currents were measured using the whole cell patch-clamp technique. Patch pipettes (3-5 M
) were filled with an internal solution containing (in mM) 35 KCl, 90 potassium gluconic acid, 10 NaCl, and 10 HEPES, with pH adjusted to 7.2 with KOH. GTP (0.5 mM) was added to
provide substrate for signal transduction pathways, and Mg-ATP (5 mM)
was included to inhibit KATP channels and provide substrate
for energy-dependent processes. Intracellular Ca2+ levels
were buffered by the addition of 10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) and 3 mM Ca2+, resulting in a free
[Ca2+]i of ~75 nM. Pipette potential and
capacitance and access resistance were electronically compensated.
Membrane currents were recorded with an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA) and under voltage-clamp mode at a holding
potential of 
60 mV. Whole cell outward currents were elicited by
applying step depolarizations in 10-mV steps from 50 to +60 mV using
pCLAMP software (Axon Instruments). Data were filtered at 5 kHz,
digitized with a Digidata 1200 analog-to-digital converter, and
analyzed with pCLAMP software. Cell capacitance was calculated from the area under the capacitive current elicited by a 10-mV hyperpolarizing pulse from a holding potential of 70 mV. Whole cell current was normalized to cell capacitance. External solutions were changed with a
multibarrel rapid-exchange system as previously described (35). All electrophysiological experiments were conducted
at room temperature.
Isolated Artery Preparation
Human proximal intrapulmonary arteries were obtained as described in Isolation of PASMCs and were sectioned into ring segments 4 mm in length. The endothelium was disrupted by gently rubbing the lumen with a cotton swab. Arterial ring segments were suspended between two stainless steel wires for isometric tension recording in tissue baths filled with Krebs bicarbonate solution containing (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 10 glucose, and 2.5 CaCl2. The solution was gassed with 16% O2-5% CO2-balance N2 at 37°C to maintain pH at 7.4. One wire was anchored in the chamber, and the other was connected to a strain gauge (model FT03; Grass Instruments, Quincy, MA) attached to a micrometer for continuous measurement of isometric tension (model 7E polygraph; Grass Instrument). The arteries were adjusted to a resting tension of 5 g in 1-g steps over a period of 45 min. Preliminary experiments revealed that contractile responses to KCl (80 mM) were maximal at this resting tension. Arteries were exposed to 80 mM KCl to establish viability and maximum contraction and to phenylephrine (3 × 107 M) followed by ACh (106 M) to verify
disruption of endothelium integrity. Vessels that did not exhibit an
increase in tension 
2 g in response to KCl or dilated by >10% in
response to ACh were discarded. To confirm that endothelial denudation
did not seriously damage smooth muscle, contractile responses to KCl in
rings subjected to denudation were required to be 80% of responses
measured in control rings with intact endothelium.
Experimental Protocols
Identification of K+ currents in
freshly isolated human pulmonary arterial myocytes.
Membrane currents were elicited by applying an 800-ms depolarizing
pulse from a holding potential of 60 mV to test potentials ranging
from 50 to +60 mV in 10-mV increments. The measurements were made
under control conditions, 3-4 min after applying charybdotoxin (ChTX; 100 nM), and 3-4 min after subsequent application of
4-aminopyridine (4-AP; 10 mM). Whole cell currents recorded in the
presence of ChTX were further characterized by analyzing the time
course of current inactivation, as described previously (35,
38), with the following biexponential equation:
I(t) = A0 + A1
1 and 2 are
the time constants of the rapidly and slowly inactivating components, respectively.
Effect of ET-1 on KV current.
To study the effect of ET-1 on the KV current, experiments
were performed in the presence of ChTX (100 nM) to inhibit
KCa currents. The effect of ET-1 on KV current
was determined by measuring peak and steady-state (at 700-800 ms)
membrane currents elicited by depolarizing pulses of 800 ms from 60
to +40 mV in 10-mV increments before and 3-4 min after application
of ET-1 (108 M).
Role of PKC in the effect of ET-1 on KV current.
The involvement of PKC activation was examined by comparing the effects
of ET-1 (108 M) on KV currents generated in
PASMCs before and 5 min after exposure to staurosporine (1 nM), a PKC
inhibitor. We have previously demonstrated that this concentration of
staurosporine is sufficient to inhibit ET-1-induced inhibition of
KV current in rat PASMCs (35).
Effect of KV channel inhibition on pulmonary arteries. To determine whether inhibition of KV channels could cause contraction in human pulmonary arteries, we examined the effect of 4-AP, a KV channel inhibitor, on baseline tension. Endothelium-denuded pulmonary arterial segments were adjusted to a baseline tension of 5 g and challenged with 1 mM 4-AP. When the increase in tension had stabilized, the arteries were then challenged with a higher concentration of 4-AP (5 mM). Contraction is expressed as maximum tension generated at each concentration normalized to the maximum tension induced by 80 mM KCl.
Role of PKC in ET-1-induced contraction.
To verify the ability of ET-1 to constrict human pulmonary vascular
smooth muscle and evaluate whether activation of PKC was involved in
the generation of tension in response to ET-1, endothelium-denuded pulmonary arterial segments were mounted for isometric tension recording. One pair of vessel segments per lung was challenged with
increasing concentrations of ET-1 (1010 to 3 × 108 M) in the absence or presence of staurosporine (100 nM) or GF-109203X (GFX; 100 nM), a specific PKC inhibitor. Contraction
was expressed as maximum tension generated at each concentration
normalized to the maximum tension induced by 80 mM KCl.
Drugs and Chemicals
ET-1 and ChTX were obtained from American Peptides (Sunnyvale, CA). GFX was obtained from Calbiochem (La Jolla, CA). Staurosporine, 4-AP, and all other chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of ET-1 (105 M) and ChTX
(104 M) were made up in distilled water, divided into
aliquots, and kept frozen at 20°C. On the day of experiment, the
solutions were diluted as needed with PSS. Stock solutions of
staurosporine (102 M), GFX (102 M), and
4-AP (101 M) were made fresh on the day of study. 4-AP
was made in PSS, and the pH was adjusted to 7.4 with HCl.
Statistical Analysis
Statistical significance was determined by the Student's t-test (paired and unpaired as applicable) and two-way ANOVA with repeated measures. A P value < 0.05 was accepted as significant. In the text, data are expressed as means ± SE, where n refers to the number of cells or arteries tested. Experiments were performed on cells from at least three different lungs or on arterial pairs from different lungs.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Identification of KV Current in Freshly Isolated Human PASMCs
Average cell capacitance of freshly isolated human PASMCs was 25.0 ± 1.66 pF (n = 21), close to values reported in cultured human PASMCs (30, 31). Outward K+ currents from human intrapulmonary arterial smooth muscle cells were measured using the whole cell voltage-clamp technique. The outward currents could be separated into two major components. One component was inhibited by ChTX (100 nM), a KCa channel antagonist. This component of current represented 17.7% of the peak and 40% of the steady-state current at +20 mV and 18.4% of the peak and 38.5% of the steady-state current at +60 mV. After inhibition of KCa channels, a second component of current was observed that exhibited rapid, voltage-dependent activation and time-dependent inactivation (Fig. 1). The current was activated at potentials positive to30 mV. 4-AP (10 mM) inhibited 85% (both the
peak and steady-state current) of the remaining ChTX-insensitive
current at +20 mV. These characteristics are consistent with the
KV current described in previous studies (35,
38).
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The inactivation kinetics of the KV current were further
quantified by fitting the descending portions of the KV
current, as described above. This approach separated the whole cell
KV current into rapidly and slowly inactivating and
noninactivating components (Table 1). All
three components contributed similarly to the total current, and all
were sensitive to 4-AP.
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Effect of ET-1 on KV Currents
After addition of 100 nM ChTX to inhibit KCa channels, application of ET-1 (108 M; n = 12)
caused a significant inhibition of KV current (Fig. 2). The current measured in the presence
of ET-1 was reduced at all test potentials positive to 30 mV. At +20
mV, ET-1 decreased peak KV current density by 29.3%, from
5.8 ± 1.5 to 4.1 ± 0.9 pA/pF, as represented in the
downward shift in the current-voltage (I-V) relationship.
ET-1 inhibited the steady-state portion of the KV current
by 48.8%, from 1.76 ± 0.4 to 0.9 ± 0.2 pA/pF. The effect
of ET-1 on KV currents in the human PASMCs was similar in
magnitude to that we previously observed in PASMCs from rats (35).
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Analysis of the KV inactivation kinetics indicated that
ET-1 significantly reduced the time constant of the rapidly
inactivating component and the amplitudes of the rapidly inactivating
and noninactivating components (Table 1). Although ET-1 also appeared
to reduce the time constant of the slowly inactivating component and
the amplitude of the slowly inactivating component, the decrease in
these values did not reach statistical significance. The effect of ET-1
on the inactivation kinetics of the KV current is shown by
superimposing the peak normalized KV current in the absence
and presence of ET-1 (Fig. 3), showing
significant enhancement of inactivation and reduction in the
steady-state current.
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Role of PKC Activation in ET-1-Induced Inhibition of KV Current
We have previously shown that PKC agonists inhibit the KV current and that ET-1-induced inhibition of the KV current in rat PASMCs requires activation of PKC (35). To determine the role of PKC activation on the effect of ET-1 on KV in human PASMCs, we pretreated PASMCs with staurosporine (109 M), a widely used nonspecific PKC
inhibitor, before ET-1 (108 M) challenge. Exposure to
staurosporine had no significant effect on KV current
(3.9 ± 0.8 to 3.6 ± 0.9 and 2.3 ± 0.8 to 2.1 ± 0.9 pA/pF for peak and steady-state current at +20 mV, respectively; n = 6). In the presence of staurosporine, ET-1 had no
effect on KV current in human PASMCs (3.5 ± 0.8 and
2.0 ± 0.8 pA/pF for peak and steady-state currents at +20
mV, respectively), causing no downward shift in the
KV current I-V relationships (Fig.
4). ET-1 also failed to decrease
KV current inactivation kinetics (Table
2), and normalized KV
currents in the presence of staurosporine before and after application
of ET-1 were identical (Fig. 5).
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Effect of 4-AP on Isolated Human Pulmonary Arteries
To determine whether an inhibition of KV channels, as occurs with ET-1, can influence tone in human pulmonary arteries, endothelium-denuded arterial segments were adjusted to a stable baseline tension of 5 g. Addition of 1 mM 4-AP caused a significant increase in tension that reached a plateau at 16.9 ± 1.2% of the maximum tension induced by 80 mM KCl (10.0 ± 2.6 g; n = 3). Subsequent challenge with 5 mM 4-AP caused an additional sustained increase in tension to 102.1 ± 8.8% of the maximum KCl tension (Fig. 6).
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Role of PKC in ET-1-Induced Contraction in Isolated Human Pulmonary Arteries
To verify the contractile effect of ET-1 on the human pulmonary vasculature, arterial segments were challenged with exogenous ET-1 while isometric tension was monitored. Application of increasing concentrations of ET-1 (1010 to 3 × 108 M) caused concentration-dependent contraction of
endothelium-denuded human pulmonary arteries (n = 4;
Fig. 7), reaching a maximum of 118.3 ± 10.3% of the tension induced by KCl at 3 × 108
M ET-1. In vessels pretreated for 15 min with staurosporine
(109 M), the ET-1-induced increase in tension was
blunted, although the difference for the entire concentration-response
curve did not reach significance with ANOVA (P = 0.07).
However, contrast analysis indicated significant differences in the
presence and absence of staurosporine at 3 × 109
and 108 M ET-1 (P < 0.01), where
contraction in response to ET-1, expressed as a fraction of maximum
tension induced by KCl, was reduced from 0.49 ± 0.05 to 0.16 ± 0.05 at 3 × 109 M ET-1 and from 1.02 ± 0.2 to 0.73 ± 0.2 at 108 M ET-1 (n = 4). Because staurosporine can have nonselective effects, the effect of
a putative selective PKC inhibitor, GFX (107 M; see Ref.
26), was also examined. At the concentration of ET-1 where
inhibition by staurosporine was maximum (3 × 109
M), GFX reduced ET-1-induced contraction, expressed as a fraction of
maximum KCl-induced tension, from 0.67 ± 0.09 to 0.37 ± 0.06 (n = 3), further supporting a role for PKC
activation in ET-1-induced contraction (Fig. 7). Maximum tension
induced by KCl, measured at the beginning of the experiment, was
similar in all arteries tested: 7.2 ± 2.1 g in control
arteries, 6.7 ± 2.0 g in arteries subsequently exposed to
staurosporine, and 9.0+1.5 g in arteries subsequently exposed to GFX.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we found that ET-1 constricts human pulmonary arteries and inhibits KV currents in human intrapulmonary arterial smooth muscle cells. Both the inhibitory effect of ET-1 on KV current and ET-1-induced contraction could be attenuated by pretreatment with staurosporine, suggesting involvement of a mechanism requiring activation of PKC.
KV channels are a major regulator of membrane potential in pulmonary vascular smooth muscle, since inhibitors of KV channels cause depolarization in rat PASMCs (2, 35, 38), increase [Ca2+]i in cultured human PASMCs (39), and increase tension in human pulmonary arteries (30). KV channels have been pharmacologically identified in cultured human PASMCs (18, 30, 39); however, because K+ channel expression and/or activity can be modified in culture (28), we verified the contribution of KV channels to the whole cell outward K+ current measured in freshly isolated human PASMCs. Under these conditions, KV current, defined as that portion of the K+ current sensitive to 4-AP but not to ChTX, comprised 57% of the total peak outward K+ current and 38% of the total steady-state K+ current. The KV current in the freshly isolated human PASMCs consisted of at least three components (noninactivating, rapidly inactivating, and slowly inactivating). These components closely resemble the KV current observed in PASMCs from the rat (35, 38) and may suggest that multiple KV channel subtypes are present.
Peng et al. (31) demonstrated a dual effect of ET-1 on
KCa currents in cultured human PASMCs, with ET-1 activating
KCa current at low concentrations and inhibiting
KCa current at high concentrations. In the present study,
we found that ET-1 also inhibited KV currents in human
PASMCs. At 108 M, ET-1 caused a 29% decrease in the peak
KV current density and a 49% decrease in the steady-state
portion of the current. The difference in magnitude of inhibition
between the peak and steady-state portions of the KV
current suggested that ET-1 might have different effects on the three
components of KV current we had identified. Comparison of
the inactivation kinetics of KV current in the absence and
presence of ET-1 indicated that, although the amplitude of all three
components appeared to decrease after addition of ET-1, the decrease in
the amplitude of the slowly inactivating component did not reach
statistical significance. In addition to decreasing the amplitudes of
the rapidly inactivating and noninactivating components, ET-1 also
decreased the time constant of the rapidly inactivating component of
the KV current. These effects of ET-1 on KV
current kinetics in human PASMCs are qualitatively similar to those we
previously reported in the rat (35).
The exact mechanism by which ET-1 inhibits KV currents is
unclear. Agonist-induced increases in [Ca2+]i
inhibit KV currents in vascular smooth muscle
(33). Because ET-1 increases
[Ca2+]i levels in rat PASMCs (4,
36), it is probable that an ET-1-induced rise in
[Ca2+]i also occurs in human PASMCs. The
inhibitory effect of ET-1 on KV currents observed in the
current study was not likely a secondary effect of increased
[Ca2+]i, however, because our cells were
dialyzed with BAPTA, a strong Ca2+ chelator that prevents
changes in global [Ca2+]i. A role for PKC
activation in ET-1-induced contractile responses has been established
in several vascular beds, including the lung (5, 11), and
studies from our laboratory and others indicate that PKC activation
inhibits KV currents in smooth muscle (1, 35).
Furthermore, we have demonstrated in rat PASMCs that the inhibitory
effect of ET-1 on KV current requires activation of PKC
secondary to activation of PLC (35). Consistent with those findings, the inhibitory effect of ET-1 on human PASMCs was abolished after pretreatment with staurosporine. The exact mechanisms involved in
PKC-induced inhibition of the KV current are not well
understood and perhaps involve direct phosphorylation of KV
channels by PKC (32), indirect phosphorylation through
second messengers that modify KV channel activation
(16), or PKC-dependent activation of 
-subunits
(20), which enhance KV channel inactivation
when associated with KV channel 
-subunits (9,
29). Further experiments will be required to elucidate the exact
mechanism responsible for this phenomenon.
Application of exogenous ET-1 to endothelium-denuded human pulmonary
arteries resulted in a concentration-dependent increase in isometric
tension. The maximum response occurred at 108 M,
consistent with results observed in the pulmonary vasculature of other
species (7, 17, 22). We previously demonstrated that ET-1
initiates a complex series of events in rat PASMCs, including
activation of PLC and PKC, inhibition of KV currents, depolarization, activation of Ca2+ influx, and
Ca2+ release from the sarcoplasmic reticulum (35,
36). An initiating step in the ET-1 contractile process in rat
PASMCs appears to be PKC-dependent inhibition of KV
currents, since depolarization preceded the rise in
[Ca2+]i (36). In the present
study, we found that 4-AP increased tension in human pulmonary arterial
segments, verifying that modulation of KV channels can
regulate tone in this vascular bed. Moreover, pretreatment of arteries
with PKC inhibitors, which prevented inhibition of KV
current by ET-1, significantly reduced the ability of ET-1 to increase
tension, providing further evidence supporting the possibility of a
role for PKC-dependent inhibition of KV current in
ET-1-induced contraction.
In summary, we found that ET-1 inhibits KV channels and causes constriction in human pulmonary arteries. Furthermore, the effects of ET-1 were attenuated in the presence of PKC inhibitors. These findings suggest that ET-1 modulates pulmonary vascular reactivity in humans, in part via signal transduction pathways involving activation of PKC and inhibition of KV currents. Moreover, the effects of ET-1 on KV channels in PASMCs from rats and humans were qualitatively similar, and both appear to require activation of PKC, suggesting that PKC-dependent inhibition of KV channels by ET-1 may be conserved among species.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-09543 (to L. A. Shimoda), HL-51912 (to J. T. Sylvester), and HL-52652 (to J. S. K. Sham) and by Scientist Development Grant AHA9930255N from the American Heart Association (to L. A. Shimoda).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. A. Shimoda, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Univ., 5501 Hopkins Bayview Circle, JHAAC 4A.52, Baltimore, MD 21224 (E-mail: shimodal{at}welch.jhu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 October 2000; accepted in final form 20 June 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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